Discoveries in ovine reproductive physiology and a new method of ....

6/17/2019

Discoveries in ovine reproductive physiology and a new method of transcervical artificial insemination for sheep

Bryan Beachy

Beachy Lincolns Farm

4264 Jefferson Marion Rd SE

Jefferson, Oregon 97352 USA

Abstract

In this multi-year study in Western Oregon, a small flock of sheep was observed to display different styles of mating behavior during coitus that were dependent on whether legumes were present in their diet. Investigation with a video probe revealed that ewes with no legumes in their diet came into heat with an open cervix, but when the probe touched the cervix, it would snap shut. A different probe, its distal end covered with foam that was saturated with ram penile mucus, did not cause the cervix to close, but easily penetrated the cervix to the uterus. Further investigation found that a sample of cervical mucus collected from the mid cervix of a ewe, when applied to the tip of a ram's extended penis, elicited a thrust and curl reflex from the ram's penis. From these findings, it is evident that phytoestrogens have greater effects on ovine cervical functionality than previously suggested; that a ewe's reproductive tract has a defense mechanism; that chemical communication occurs between a ram's penis and a ewe's cervix; that in an optimal mating the ram's penis penetrates the ewe's cervix and ejaculates in her uterus; that in a mating involving a ewe with phytoestrogen induced closed cervix syndrome the ram ejaculates in the vagina; that the functional state of a ewe's cervix is readily apparent by observation of mating behavior; that by limiting phytoestrogens in ewes' diets and mimicking sheep's natural processes, transcervical artificial insemination is a simple procedure; and that a better method of collecting ram semen would involve the use of an artificial cervix filled with synthetic cervical mucus.

Keywords

Sheep; Reproductive physiology; Phytoestrogen; Artificial insemination; Semen collection.

1. Introduction

This study was initiated for the purpose of developing a simple, effective method of transcervical artificial insemination for sheep that would enable sheep producers to enjoy the benefits of an AI industry like that enjoyed by cattle producers.

The first 27 years of this study were spent making observations of a small(40 to 60 hd) flock of Romeldale sheep in Western Oregon environments that included: one permanent winter quarters with pasture of mixed grasses and 5 to 10% clover, where alfalfa hay was fed at 4 to 5 lbs/hd/day; and three different spring through fall pastures, two of which contained legumes, and one of which contained no legumes. Breeding dates, mating styles, lambing dates, conception rates, and lambing percentages were recorded. During that time, three distinct styles of mating employed by rams during the act of coitus were observed and noted.

Style #1, "beginning heat style", involved the ram mounting the ewe, making shallow or zero penetration of the vagina, and exhibiting a curled glans while making very rapid, short thrusting motions and jerking and skipping his hind feet, driving himself into an overly upright stance that frequently culminated in the ram falling on his back. This mating style was only observed in the years when rams were turned out early in an attempt to back up the lambing season, and only in a portion of the flock, and only on those ewe's first heats of the season. No environmental factor seemed to be involved. Since no conceptions were ever recorded from this mating style, its causes were not investigated.

Style #2, "closed cervix heat style", involved the ram mounting the ewe, making deep penetration of the vagina with a series of three to six thrusts, the last being the deepest and held frozen in place for two to four seconds, then dismounting and upon withdrawal, the penis exhibiting a curled glans. This mating style was observed on the years when the ewes spent the spring, summer, and fall on pastures containing mixed grasses and legumes, with the legumes consisting of white clover, vetch, and trefoil as 5 to 10% of the pasture. This mating style resulted in 90 to 99% conception rates, and 150 to 175% lambing rates.

Style #3, "optimal heat style", involved the ram mounting the ewe, making deep penetration of the vagina, as part of the mount, then shifting his hindquarters sideways from one hind foot to the other hind foot two to five times, then making a single forward thrust and holding it frozen in place for three to five seconds, then dismounting and upon withdrawal, the penis exhibiting a straight glans. This style of mating was observed on the years when the ewes spent the spring, summer, and fall on a pasture that was a pure stand of mixed grasses with zero legumes. This mating style resulted in 98 to 100% conception rates, and 200 to 220% lambing rates.

The difference in productivity between Style #2 matings and Style #3 matings was consistent with Davenport's findings where his phytoestrogen free(or nearly so) ewes' higher level of productivity was largely due to their much higher level of twinning ([1]Davenport,1967).

Much has been written about the difficulty of passing an AI instrument through the closed, or tortuous cervix of a ewe in heat ([2]Fair et al., 2005; [3]Halbert et al., 1990; [4]King et al., 2004). Also, the physical damage done to the ovine cervix by phytoestrogen poisoning has been well documented ([5]Adams, 1990; [6]Lightfoot and Adams, 1979). Lightfoot suggested the functional ability of the ovine cervix was impaired by phytoestrogen poisoning ([7]Lightfoot et al., 1967). So, when the different mating styles in this study were found to be dependent on the presence or absence of legumes in the ewes' diets, the suspicion arose that phytoestrogens may have greater effects on ovine cervical functionality than previously known. It was decided to begin investigations with an examination of the cervical state of estrous ewes that had been raised and maintained in the environmental conditions that gave rise to Mating Style #3.

2. Materials and methods

In preparation for the investigational phase of this study, the flock was moved to a different Western Oregon farm where the grazing fields had been sown to ryegrass and orchardgrass, and sprayed annually to eliminate weeds and legumes. Also, the flock was reorganized at this time. The aging Romeldale flock was culled down to six of the better ewes which made Group A. These were ewes that grew up under limited legume consumption management, with zero legume consumption from spring through fall. Twenty Lincoln ewes were purchased from a commercial producer to form Group B. These were ewes that grew up under limited legume consumption management year round. Ten Lincoln ewes and two Lincoln rams were purchased from farms where legumes were consumed at a moderate to heavy rate year round to form Group C. The flock was then put through a year of breeding and lambing with winter feeding of zero legume grass pasture supplemented with third cutting alfalfa hay at the rate of four to five pounds per head per day; and spring, summer, fall grazing on zero legume grass pasture. At weaning time all ewes that had production problems were culled, leaving a flock of six Romeldales for Group A, ten Lincolns for Group B, four Lincolns for Group C, and one vasectomized Lincoln ram to serve as a teaser. When the investigations commenced in September, the ewes diet had consisted of nothing but zero legume pasture for five months. Free choice sheep mineral and fresh water were available year round.

A shallow blind alley(chute) was constructed in the barn's central working pen for restraining ewes while examining, and a 12' by 12' pen was set up adjacent to the working pen to house a teaser ram, and built with cattle corral panels so the ram could tease the ewes through the bars.

The ewes were confined in the barn at night, and released to graze in the daytime. After evening lockup, and before morning turnout, the teaser ram was allowed out of his pen for 30 minutes to heat detect the ewes. No heat regulating products were employed; all heats were natural.

2.1. Viewing probe investigations

A vaginal viewing probe was constructed by gluing two 16 mm by 150 mm plastic test tubes open end to open end, heat forming the distal end into a taper with a 4 mm hole drilled in the tip, and cutting off the opposing end for camera access. Inserting the camera end of a 3.9 mm flex shaft video borescope into the probe's open end to within a few millimeters of the 4 mm hole in the distal tip afforded on screen viewing.

2.1.1. Viewing probe, in the chute.

Four Group A Romeldales were examined. As each ewe came into heat, she was penned, haltered, led(or pushed) into the chute, tied there, and scoped with the viewing probe. In the first three ewes, an attempt was made to pass the viewing probe through the cervix. Ram mating style, vaginal/cervical characteristics, and cervix and ewe's reactions were noted.

2.1.2. Viewing probe and specula, in the chute.

Two Group A ewes, six Group B ewes, and two Group C ewes were examined. As each ewe came into heat, she was penned, haltered, led(or pushed) into the chute, tied there, and scoped with the viewing probe. Then the probe was withdrawn and the ewe reexamined alternately with a duckbill or acrylic tube speculum. Ram mating style, vaginal/cervical characteristics, and cervix and ewe's reactions were noted.

2.1.3. Viewing probe in a shrink pen.

Three Group A ewes, four Group B ewes, and three Group C ewes were examined, using a different method of capture. As each ewe(or ewes) came into heat, the teaser ram was haltered and led into the working pen with the ewe(or ewes) following. The ram was then tied in the pen, the pen closed on both(or more) sheep and shrunk, and the ewe/ewes haltered and tied next to the ram. The ewe was then scoped with the viewing probe. Ram mating style, vaginal/cervical characteristics, and cervix and ewe's reactions were noted.

2.2. Transcervical probe investigation

A different probe was constructed of stainless steel tubing 36 cm long by 10 mm diameter, shaped to a curve with a radius of 50 cm; the distal end tapering 3 cm to an 8 mm dia. rounded tip, and covered for 10 cm with a 1 mm thick layer of semi absorbent foam. The tip was bored for a 4 mm hole. The caudal end was flared to accept a small test tube stopper.

A small(000) rubber test tube stopper was bored with a 4 mm hole, and the flex shaft camera inserted through that hole and made fast at the point where, when inserted in the probe, the test tube stopper secured in the probe's flared caudal end and the camera lens came to within 6 mm of the 4 mm opening in the probe's distal end. This kept the camera an adequate distance from the opening to prevent lens contamination, yet close enough to the opening for an acceptable field of view. This also stopped airflow through the probe, preventing lens contamination.

An Ovine-Caprine semen syringe(IMV Technologies) for use with 1/4 cc straws, was disassembled and rebuilt to be the length of a standard AI gun for cattle, and curved to a radius of 50 cm. Two Ovine-Caprine sheaths(IMV Technologies) glued end to end made acceptable length sheaths. Each sheath was marked bright red 37 cm from its distal tip as a guide to show when it protruded 1 cm beyond the probe's tip.

All of the ewes from Groups A and B were examined with this probe. The shrink pen and halters restraint method noted in 2.1.3. was used. To examine a ewe, the teaser ram was untied and allowed to mount the ewe, his penis deflected, and the probe's foam end wiped on the penis to coat the foam with mucus. The ram's penis was then released, and while the ram was pacifying himself with the ewe, the gloved hand that had deflected the penis was used to evenly distribute and saturate the penile mucus onto/into the foam on the distal end of the probe. The ram was then retied to the pen wall, the camera inserted into the probe with the stopper plugged tightly into the caudal flare, downward pressure exerted on the ewe's tailhead with the heel of the gloved hand with thumb and forefinger extending downward to part the vulva, the probe inserted, and the ewe examined. When the probe reached the uterus, the gloved hand was moved from the tailhead to grasp the probe's caudal end in a fist with the little finger pressed against the vulva, to lock the probe in place. The stopper was then wiggled loose, and the flex shaft retracted 7 to 8 cm and held there with the gloved hand thumb and forefinger. The AI gun was then inserted up to the red mark and the plunger depressed. The AI gun, then the probe, were withdrawn. Ram mating style, vaginal/cervical characteristics, and cervix and ewe's reactions were noted.

2.3. Cervical mucus to penis investigation

A swab of sufficient length to reach through the transcervical probe was made by cutting one end off a cotton swab and tightly inserting the swab's shaft into a tapered hole drilled in the end of a bovine insemination pipette.

An estrous ewe from Group B was probed with the transcervical probe into her cervix to the third cervical ring. The long handled swab was then inserted through the probe, into the cervix, where a sample of cervical mucus was swabbed from mid-cervix. The swab, then the probe, were withdrawn. The ewe's reactions were noted.

The teaser ram was then allowed to mount the ewe, his penis deflected, and the mucus saturated swab was applied to the tip of the ram's penis. The ram's reactions were noted.

3. Results

3.1. Viewing probe investigations

3.1.1. Viewing probe, in the chute

All four Group A ewes mated with the teaser ram in the #3 optimal style. The first three Group A ewes were calm and agreeable about being haltered and led into the chute, and exhibited a vagina free of mucus, with small dark pink bumps on the floor of the vagina surrounding the cervix, and a cervix open 4 to 5 mm filled with mucus in the form of transparent bubbles; and when the probe touched the cervix, the cervix snapped shut. The fourth Group A ewe was disagreeable about being haltered and pushed into the chute, and exhibited a vagina free of mucus, with small dark pink bumps on the floor of the vagina surrounding the cervix, and a closed cervix.

3.1.2. Viewing probe and specula, in the chute.

Both Group A ewes, and all six Group B ewes mated with the teaser ram in the #3 optimal style. One Group A ewe, and three Group B ewes were calm and agreeable about being haltered and led into the chute; and examined with the viewing probe, exhibited a vagina free of mucus, with small dark pink bumps on the floor of the vagina surrounding the cervix, and a cervix open 4 to 5 mm filled with mucus in the form of transparent bubbles; but when reexamined with either speculum, exhibited a vagina free of mucus, with small dark pink bumps on the floor of the vagina surrounding the cervix, and a closed cervix. The other Group A ewe and three Group B ewes were disagreeable about being haltered and pushed into the chute; and when examined, both the viewing probe and specula revealed a vagina free of mucus, with small dark pink bumps on the floor of the vagina surrounding the cervix, and a closed cervix.

Both Group C ewes mated with the teaser ram in the #2 closed cervix style. One Group C ewe was calm and agreeable about being haltered and led into the chute. The other Group C ewe was disagreeable about being haltered and pushed into the chute. Both Group C ewes, when examined with both the viewing probe and specula, exhibited a vagina with much mucus and no vaginal floor bumps, and a closed cervix.

3.1.3. Viewing probe in a shrink pen.

All three Group A ewes and all four Group B ewes mated with the teaser ram in the #3 optimal style. All three Group A ewes and all four Group B ewes were calm and agreeable about being haltered and tied next to the ram. All three Group A ewes and all four Group B ewes, when examined with the viewing probe, exhibited the vaginal and cervical state typical of a ewe with an open cervix.

All three Group C ewes mated with the teaser ram in the #2 closed cervix style. All three Group C ewes were calm and agreeable about being haltered and tied next to the ram. All three Group C ewes, when examined with the viewing probe, exhibited a vagina with much mucus and no vaginal floor bumps, and a closed cervix.

3.2. Transcervical probe investigation

All of the Group A and Group B ewes mated with the teaser ram in the #3 optimal style. All of these ewes were agreeable to being handled with the shrink pen and halter system. All of these ewes exhibited the vaginal and cervical state typical of a ewe with an open cervix. None of these ewes closed her cervix when it was touched with the probe; rather each cervix remained open, pliable, and easily traversed to the uterus. In each case: when the probe tip touched the cervical opening, a curtain of transparent cervical mucus adhered to the probe tip, covering the 4 mm hole, causing only a slight loss of visibility; the openings of the rings were aligned in a straight line; the cervix was filled with transparent mucus bubbles; the cervix contained six rings; and upon reaching the opening of the sixth ring the uterus beyond appeared as a large, dark, empty space. There were no adverse reactions to the insertion of the AI gun.

3.3. Cervical mucus to penis investigation

The Group B ewe made no reaction to the swab contacting her mid cervical rings, and the cervix did not close. When the cervical mucus saturated swab was applied to the tip of the ram's penis, the ram made a single thrust motion, involving both a back hump and an extension of the penis. Immediately following the extension, the tip of the penis curled down and back, in a "U" shape, and continued in that motion, with the tip of the penis moving along the underside of the penis shaft, until the entire glans was inverted 180 degrees. The filiform appendage remained limp, and the ram did not ejaculate.

4. Discussion

4.1. Clover damage

With the exception of the fields that had been purposely sprayed to eliminate legumes, the one legume common to, and most abundant in, all of the fields in which this flock was observed was Dutch White Clover. Although the legume percentages in the fields were noted, in actual practice these percentages made little difference. What mattered was time of year, and if there were legumes present, or if there were not. This was due to the sheep's grazing habits. Beginning in early to mid summer, when the weather warmed and the grasses began to mature, the sheep began to exhibit a marked preference for clover in their diet, and they would trample and waste much grass to find it. By contrast, sheep at winter quarters tended to avoid clover, and kept the pasture grasses nibbled short. Samples of pasture clover and alfalfa hay were sent to North Dakota State University Veterinary Diagnostic Laboratory for phytoestrogen screening. The hay was found to contain coumestrol at 37 ppm. Used as limited, winter only feed, it caused no harm to the ewes' breeding season cervical functionality. The clover was found to contain a low concentration of biochanin A at 100 ppm, and a low to moderate concentration of formononetin at 373 ppm. Available, and mostly uneaten, in winter only pasture, it caused no harm to the ewes' breeding season cervical functionality; but available, and eagerly consumed, in summer pasture, it appears to be a likely cause of closed cervix syndrome and associated lowered conception and lambing rates. Aside from pointing an accusing finger at White Dutch Clover in summer grazing, this study raises more questions than it answers. Sheep producers are going to want to know: which legume species are harmful to ewes' cervical function?, at what times of year?, in what amounts?, what would make better forages?, etc. Unfortunately, all of these questions are beyond the scope of this project. Further study is warranted in this area.

Five months of legume free grazing prior to breeding season was sufficient to clear the phytoestrogens from the reproductive tracts of Group A and Group B ewes, allowing optimal cervical function. This was not the case with the Group C ewes. Whether this was because the Group C ewes, with their history of moderate to heavy legume consumption, needed a longer period of zero legume diet to clear their heavier load of phytoestrogen from their reproductive tracts, or because their heavier load of phytoestrogen had caused permanent damage remains unclear; however, the permanent infertility described by Adams seems the most likely cause ([5]Adams, 1990).

4.2. Ram response to cervical mucus

When a ewe suffering closed cervix syndrome comes into heat, her dysfunctional cervix cannot contain the cervical mucus, and the mucus spills out, into the vagina. When a ram breeds such a ewe, he extends his penis into the vagina, where making contact with cervical mucus, his penile sexual reflexes are triggered simultaneously, causing him to ejaculate in the vagina, and display the #2 mating style. When a ewe in optimal reproductive health comes into heat, her dilated cervix contains all of the bubbly cervical mucus, and the vagina is mucus free. When a ram breeds such a ewe, he extends his penis into the vagina, searches for the cervix, thrusts into the cervix, and then ejaculates into the uterus; displaying the #3 mating style. This has his sexual reflexes being triggered in sequence. This sequencing of a ram's sexual reflexes may be of importance to the collection of ram semen. One problem with the use of an artificial vagina(AV) is the AV's lack of the proper chemical stimuli. A better method of collecting semen might involve the use of an artificial cervix, filled with synthetic cervical mucus bubbles, between the AV and the collection cone. Ejaculating a ram with the proper chemical stimuli in the proper sequence might improve his volume/quality of ejaculate. It would also be useful to find if the scent of cervical mucus could elicit penis extension in a ram, or induce a ram to mount a dummy ewe.

4.3. Ewe defense mechanism

The species defense mechanism of a ewe has a number of activation triggers, and defensive responses. This study identified several of these, however there may be more. One response not mentioned elsewhere is the ewe's ability to rid herself of a foreign object that has managed to penetrate her cervix. One of the early generation transcervical probes used in this study is worth mention due to the manner of its failure. This probe was mistakenly constructed with the foam covering its distal end a few millimeters shorter than the length of a ewe's cervix. In use, when the probe tip reached the uterus, the first ring of the cervix made contact with the probe's bare shaft, triggering the defense mechanism. Unable to close her cervix, the ewe erupted in a violent jack hammering motion that sent instruments flying in all directions. Another possible defense response would be if the cervix has the ability to release spermicidal compounds. Various trans/intra-cervical AI procedures in sheep have had limited success, but the one procedure, the Gourley ScopeTM, capable of depositing semen in the uterus resulted in zero pregnancies([8]Riese, 1998). This seems counterintuitive, until consideration is given to the four to five minutes required for the Gourley ScopeTM to penetrate the cervix, and during that time, the scope was stimulating the defense mechanism's receptors located in the first ring of the cervix. The possibility of spermicidal compounds in a ewe's cervical mucus is of importance to anyone collecting cervical mucus samples for analysis and synthesization. Great care should be exercised to avoid triggering the defense mechanism when collecting such samples, because, as the use of synthetic cervical mucus would be for the collection of semen, engineering spermicides into that mucus would be detrimental.

4.4. Transcervical probe and sheep handling

The transcervical probe used in this study was functional, but the need to clean it between uses, and having to wipe it on a ram's penis prior to use, make it less than desirable for commercial production and use. It would be much improved by eliminating the foam from its distal end, and giving it single use plastic sleeves which have foam on their distal ends and are pre-saturated with synthetic ram penile mucus. Learning how to use this probe is easy, as the vaginal floor bumps surrounding the cervix provide a visual aid in locating the cervix; but learning how to handle ewes in such a manner that their cervixes remain open and receptive is more of a challenge: probably the biggest challenge facing the future ovine AI industry. Although the shrink pen and halters method used in this study was effective, that method is not ideal for several reasons: it involves the use of a vasectomized teaser ram(inviting disease transmission and aggressive ram behavior), using halters is cumbersome, and it doesn't do enough to teach the ewes to transfer their libido attachments to a human technician. A ewe's acceptance of a human as a ram surrogate is possible, as demonstrated by the behavior of two of the ewes in this flock. After being transcervically probed a few times their in heat behavior during heat detecting changed. Upon teaser ram turnout they would trot right past the ram, approach the investigator, lower their heads, and push against the investigator's ankles while wagging their tails. When the investigator walked around to their hindquarters, swiped a boot down the outside of their hind leg and applied knee pressure to their rump while making chuckling ram noises, they would spread their front legs and brace themselves, then hold perfectly still while the investigator crouched behind them and partially probed them. Had a supply of synthetic ram penile mucus been available, they could have been AI bred that way: with no pen, no halter, and no ram. The behavior of these two ewes holds real possibilities for the future of AI in sheep. An estrous ewe will frequently put her head under the ram's belly and sniff at the ram's prepuce. If part of that behavior is scent recognition, and if the scent involved is from the ram's penile mucus, then an AI technician might make fruitful use of synthetic penile mucus in a scent emitter strapped to his/her lower leg to identify the technician to the ewes as a surrogate ram. Better methods of ewe handling need to be identified.

3.5. Other species

Given the similarities between sheep and goats in reproductive physiology, it seems reasonable to suspect that much of the information gained from this study may apply to goats as well; with the exceptions that goats don't seem to have a defense mechanism, and the chemical signatures of their mucus should be different than those of sheep. Although not strictly necessary, the use of the methods in this study might encourage the wider use of AI in goats for humane reasons.

Acknowledgements

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

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Declaration of interest:

The author(Bryan Beachy) has filed patent applications for the transcervical probe, modified AI gun, and artificial cervix discussed in this manuscript.